The goal of this proposal is to test the hypothesis that the fully mature mammalian spermatozoon has a limited capacity to manipulate its chromatin in response to its environment, providing a mechanism to prevent the propagation of DNA that has been damaged during the life of the cell. Specifically, we propose that the spermatozoon can undergo a type of apoptotic DNA degradation, and a consequence of this ability is that the sperm cell also interacts with exogenous DNA. This has significant implications for research in many areas such as the manipulation of spermatozoa for in vitro fertilization (IVF), the development of male contraceptives, sexual transmission of diseases, the production of transgenic animals, and gene therapy. We have proposed a model for sperm chromatin structure that provides a possible mechanism for the spermatozoa-specific apoptosis and interaction with exogenous DNA, in which the protamine bound DNA toroids are linked by histone bound "spacer" DNA segments. We propose that these histone bound spacers, which we term active chromatin foci, are the sites where apoptotic DNA degradation takes place, and the sites of sperm chromatin interaction with exogenous DNA. We will test the specific hypothesis that mammalian spermatozoa have the ability to undergo a specific type of apoptotic DNA degradation and can interact with DNA, and that these activities occur at the histone bound active chromatin foci. We will first determine the distribution of histone bound DNA by pulse-field gel electrophoresis and Southern blot analysis, then compare this distribution to the apoptotic DNA degradation pattern. We will then determine how mouse spermatozoa interact with exogenous DNA. Our preliminary data suggests that live spermatozoa bind to and incorporate exogenous DNA onto a specific region of the nuclear matrix. We will determine whether this DNA is integrated into the sperm genome. Finally, we will isolate and characterize a topoisomerase II- like protein that we have identified in hamster sperm nuclear matrices. This is an enzyme that might be expected to be involved in both apoptosis, DNA binding, and integration. This application represents a new direction for our laboratory that will lead us to focus on therapeutic and contraceptive studies.